Our long term goal is to study the mechanism of neurodegeneration induced by environmental neurotoxicants. This proposal is submitted to investigate the active role of astrocytes in regulating the levels of environmental neurotoxic cations and hence, in modulating neurodegeneration. Based on our preliminary data we hypothesize that cations such as MPP+ (1-methyl-4-phenylpyridinium) and paraquat (PQ) are bi- directionally transported across the astrocytic plasma membrane by the organic cation transporter 3 (OCT3) and, through this mechanism, OCT3 modulates neurotoxicity. Thus, the tissue and cellular distribution of OCT3 should be critical in defining the differential regional susceptibility to cationic neurotoxins. Cations representing two different categories of environmental neurotoxicants with different toxicokinetics will be used. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a lipophillic compound that will be used to generate MPP+ inside of astrocytes. The goal is to assess how the release of MPP+ from astrocytes (v/a OCT3) into the extracellular space would subsequently induce selective death in the nigral dopaminergic neurons. PQ, a widely used cationic herbicide that has been linked to parkinsonism, will be used to assess how astrocytes affect neurodegeneration by taking up (via OCT3) and thus removing toxic cations from the extracellular space. Of note, both MPP+ and PQ also increase the outflow of the endogenous cation dopamine (DA), which is neurotoxic upon oxidation To test our hypotheses, mutant mice deficient in OCT3 and an OCT3 inhibitor will be used. In the first specific aim, we will assess how OCT3 regulates the levels of MPP+, PQ and DA by determining its uptake and reverse transport kinetics for these cations using both cell culture and animal models. In the second specific aim, we will evaluate how OCT3 modulates neurotoxicity through its bi-directional transport of MPP+ and PQ. We hypothesize that OCT3 ablation, by sequestrating MPP+ in astrocytes, attenuates dopaminergic neuronal death after MPTP treatment. Conversely, OCT3 ablation, by preventing the uptake of MPP+, PQ, and DA into astrocytes, enhances dopaminergic neuronal death after MPP+ and PQ treatments. Thus, our plan is to assess the magnitude of dopaminergic neurotoxicity in OCT3 mutant mice as well as co-culture models of astrocytes and dopaminergic neurons, treated with MPTP, MPP+ or PQ. We will also assess whether re-expression of OCT3 in astrocytes deficient in this transporter would reverse the neurotoxic effects. The proposed studies have potential to unravel a still unrecognized pathway by which different cell types in the brain interact with each other to modulate neurodegeneration induced by environmental toxicants. In addition, these studies may provide significant insights into a novel mechanism that contributes to the pattern of cell death as seen in neurodegenerative disorders such as sporadic Parkinson's disease.